Bioactive derivatives of camptothecin

ABSTRACT

Water soluble polymeric conjugates of camptothecin consist essentially of N-(2-hydroxypropyl)methacryloylamide units linked via a spacer group to a residue of a camptothecin such as irinotecan or its non-soluble metabolite, 7-ethyl- 10 -hydroxy amptothecin. The conjugates possess enhance antitumor activity and decreased toxicity with respect to the free drug. A process for their preparation and the pharmaceutical compositions containing them are also described.

[0001] The present invention provides water soluble polymeric conjugates of camptothecin possessing enhanced antitumor activity and decreased toxicity with respect to the free drug. 7-Ethyl-10-hydroxy-camptothecin (1a, Z=H), a topoisomerase I inhibitor belonging to the class of camptothecin, is a non-soluble compound and it is recognized as the active metabolite of irinotecan (CPT- 11, 1b, Z=1,4′-bipiperidinecarbonyl, Cancer Res.: 50, 1715-20, 1990).

[0002] The invention provides a polymeric conjugate which is denoted herein as (A) and which consists essentially of:

[0003] (i) from 85 to 97 mol % of N-(2-hydroxypropyl)methacryloylamide units represented by formula (2)

[0004] (ii) from 3 to 15 mol % of units represented by formula (3)

[0005] wherein [S] is a spacer group; Z is hydrogen, R₁CO in which R₁ is C₁-C₆ alkyl or the group 1,4′-bipiperidine and

[0006] (iii) from 0 to 12 mol % of N-methacryloyl-glycine or N-(2-hydroxypropyl) methacryloyl-glycinamide units represented by formula (4)

[0007] wherein [X] represents hydroxy or a residue of formula —NH—CH₂—CH(OH)—CH₃. The polymer of the present invention may also be represented as follows: [(2)]_(x); [(3)]_(y); [(4)]_(z) wherein (2), (3) and (4) are units of the formula as above defined, and x is from 85 to 97 mol %, y is from 3 to 15 mol % and z is from 0 to 12 mol %. Preferably, the polymeric conjugate (A) contains the N-(2-hydroxypropyl) methacryloyl amide units represented by the formula (2) in a proportion of 90 % or more; more preferably 90%. The conjugate may also contain from 3 to 10 mol% of the 20-O-(methacryloyl-glycyl-peptidyl)camptothecin units represented by the formula (3), more preferably 10 mol % of such units. Preferably A does not contain residues of formula (4), i.e.zis0.

[0008] The spacer group is susceptible to intratumoral enzymatic hydrolysis. The spacer group may be an amino acid residue or a peptide spacer, for example from 2 to 4 amino acid residues long. Preferably the spacer group [S] is selected from Ala-Gly, Phe-Gly, Leu-Gly, Phe-Ala, Leu-Leu, Phe-Leu-Gly, Leu-Leu-Gly and Phe-Leu-Gly-Gly.

[0009] Preferably, the polymeric conjugate (A) contains the units represented by formula (3) wherein Z is H.

[0010] Content of active camptothecin derivative of formula (1) in the conjugate of formula A may be from 2 to 15% (weight/weight), more preferably 10% (w/w). The invention also provides a process for preparing a polymeric conjugate as defined above, which process comprises reacting a camptothecin derivative of formula (5):

[0011] wherein [S] and Z are as defined above, with a polymer (B) consisting essentially of:

[0012] (i) from 85 to 97 mol % of N-(2-hydroxypropyl)methacryloylamide units represented by formula (2)

[0013] (iv) from 3 to 15 mol % of N-methacryloyl-glycyl units represented by formula (6)

[0014] wherein R₂ is

[0015] the residue of an active ester, or

[0016] hydroxy;

[0017] and optionally displacing the remaining active ester groups with 1-amino-2-propanol.

[0018] The invention also provides 20-O-peptidyl-camptothecin derivatives of formula (5′)

[0019] wherein the group [S′] is a peptide spacer, for example from 2 to 4 amino acid residues long, preferably selected from Ala-Gly, Phe-Gly, Leu-Gly, Phe-Ala, Leu-Leu, Phe-Leu-Gly, Leu-Leu-Gly and Phe-Leu-Gly-Gly; Z is as defined above, and their salt derivatives. Such compounds are prepared by condensing camptothecin derivatives of formula 1 as above defined with a N-protected-peptidyl derivative of formula (7):

R ₃—[S′]—P   (7)

[0020] wherein [S′] is as defined above and R₃ represents an amino-protecting group, such as Boc, FMOC, triphenylsilyl, diphenylmethylene or triphenylmethyl, and P is a residue of an activated ester, such as p-nitrophenoxy or N-hydroxysuccinimido, to give a compound represented by formula (8):

[0021] wherein Z′ is R₃[S′] or R₁CO, wherein R₁, R₃ and [S′] are as defined above and then deblocking the N-protecting group of the substituent at position C-20 from the resulting compound. The compounds of formula (5) as defined above may be analogously prepared.

[0022] Preparation of compounds of formula (7) follows standard synthetic procedures that are known from the literature. Suitable N-protected-peptidyl derivatives of formula 7 include, for example, N-(tri-phenylmethyl)-L-phenylalanyl-L-leucyl-glycyl p-nitrophenylester (7a), N-(t-Butoxycarbonyl)-L-phenyl-alanyl-L-leucyl-glycyl p-nitrophenylester (7b). Thus, for example, 7-ethyl-10-hydroxy-camptothecin (1a) may be allowed to react with a molar excess, for example up to five-fold molar excess or more, especially 2 mol.equivalent, of a N-protected-peptidyl derivative of formula (7) in anhydrous solvent such as dry dimethylformamide in the presence of 4-dimethylaminopyridine. In this manner, the protected amino acid is introduced at both hydroxylated positions 10—OH and 20—O of compound 1a to give compound of formula (8a: Z=R ₃ [S′]).

[0023] The reaction can typically be effected for from 8 to 48 hours. The reaction is typically carried out at a temperature from 15 to 40° C. The substituent group at position 10—OH and the amino-protected group R₃ of the substituent at 20—OH are removed by an appropriate deprotecting agent to give the 7-ethyl-10-hydroxy-20-O-peptidyl-camptothecin derivative of formula (5a: Z=H). Deprotection may be therefore achieved by acid treatment, such as treatment with acetic acid, a mixture of acetic acid and 1.5N aqueous hydrochloric acid or 90% aqueous trifluoroacetic acid from one to 6 hours at temperature from 10 to 30° C.; preferably for two hours at room temperature.

[0024] The condensation of derivative of formula (5) with the polymer of formula (B), is carried out in conditions capable of preserving the nature of linkage between 7-ethyl-10-(substituted)-camptothecin and spacer [S] as well as that of the conjugate.

[0025] Polymers consisting essentially of from 85 to 97 mol % of N-(2-hydroxypropyl)-methacryloylamide units of formula (2) and from 15 to 3 mol % of N-methacryloyl-glycine units of formula (6), are prepared by copolymerization of N-(2-hydroxypropyl) methacrylamide with N-methacryloyl-glycine or methacryloyl-glycine active-ester derivatives, as described in Makromol.Chem. 178, 2159 (1977). R₂ may represent a phenoxy group which is substituted on the phenyl ring by one ore more electron-withdrawing groups, such as nitro or halogen. Preferably R₂ represent p-nitrophenoxy. Preferably, the reaction between a polymer in which R₂ represents the residue of active ester and a compound of formula (5) to prepare the polymeric conjugate of the invention is carried out in an anhydrous polar organic solvent such as dimethylsulfoxide. The reaction can typically carried out at temperature from 15 to 30° C., preferably at room temperature for 15 hours; then the aminolysis of the remaining active ester groups can be performed in the presence of 1-amino-2-propanol at room temperature, from 0.5 to 1 hour. The conjugate suitably is precipitate with ethyl acetate, dissolved in ethanol and reprecipitated with ethyl acetate.

[0026] For example, the polymer in which R₂ represents the residue of an active ester, provided at a concentration of 15% (w/v) in dry dimethylsulfoxide, is treated with 7-ethyl-10-hydroxy-20-O-peptidyl-camptothecin derivative of formula (5a), 3% (w/v), at room temperature for 15 hours. Then 1-amino-2-propanol, 0.1% (w/v) is added and the reaction mixture is kept at room temperature for 1 hour. The conjugate can be precipitated with ethyl acetate, collected, washed with ethyl acetate, then dissolved with absolute ethanol at a concentration of 10% (w/v) and precipitated again with ethyl acetate to give the conjugate of formula (A) according to the invention.

[0027] The content of active drug (1), in polymeric conjugate of the invention is determined by HPLC or absorbance spectroscopy analysis.

[0028] The polymer-bound derivatives of formula (A) are in the range of 5.000 to 45.000 molecular weight, preferably from 18.000 to 35.000. Polymeric drug derivatives of formula (A) are water soluble and show enhanced antitumor activity and reduced toxicity in comparison with the free 7-ethyl-10-hydroxy-camptothecin. They are useful in the treatment of leukemia and solid tumors, such as colon, colo-rectal, ovarian, mammary, prostate, lung, kidney and also melanoma tumors. A human can therefore be treated by a method comprising administering thereto a therapeutically effective amount of a polymeric conjugate of the invention. The condition of the human patient can thus be improved.

[0029] The dosage range adopted will depend on the route of administration and on the age, weight and condition of the patient being treated. The polymeric conjugates of formula (A) as well as soluble salt derivatives of formula (5′) are typically administered by parenteral route, for example intramuscularly, intravenously or by bolus infusion. A suitable dose range is from 1 to 1000 mg of equivalent per m² body surface area of active drug of formula (1), for instance from 10 to 100 mg/m².

[0030] The polymeric conjugate (A) or soluble salt derivatives of formula (5′) may be formulated into a pharmaceutical composition together with a pharmaceutically carrier or diluent. Typically they are formulated for parenteral administration, for example by dissolution in water for injection or physiological saline.

[0031] The following Examples illustrate the invention.

[0032] Example 1.

[0033] Preparation of: 7-ethyl-10-hydroxy 20-O-(Lmphenylalanyl-L-leucyl-glycyl)camptothecin (5c; H[S] =H-Phe-Leu-Gly-, Z≈H)

[0034]7-ethyl-10-hydroxy-camptothecin (1a, 0.8g, 2mmol), ), N-(tert-Butoxycarbonyl)-L-phenyl-alanyl-L-leucyl-glycyl p-nitrophenylester (7b; 3.3g, 6mmol) and 4-dimethylaminopyridine (0.5g, 4mmol) were dissolved with dry dimethylsulfoxide (30ml) and kept at room temperature for three days under stirring. After that the reaction mixture was poured in 0.1N aqueous hydrochloric acid (500ml) to give a precipitate which was collected on a sintered glass funnel. The solid material was dissolved in ethyl acetate (200ml) and washed with saturated aqueous solution of sodium hydrogen carbonate (2×100ml) and water (2×100ml). The organic phase was separated, dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue was treated with 90% aqueous trifluoroacetic acid (40ml) for three hours, then the solvent was removed under reduced pressure. The crude material was dissolved in methanol (50ml), diluted with methylene chloride (200ml), washed with saturated solution of sodium hydrogen carbonate (3×100ml) and water (3×100ml). The organic phase was dried over anhydrous sodium sulfate evaporated under reduced pressure and flash chromatographed on silica gel using a mixture of methylene chloride/methanol (95/5 v/v) as eluting system to give 0.93g of the title compound (5c).

[0035] TLC on Kieselgel plate F₂₅₄ (Merck), eluting system methylene chloride/methanol (95/5 v/v), R_(f=)0.18.

[0036]¹H-NMR (200 MHz, DMSO) 0.80 (d, J=5.7Hz, 6H, δ+δ′-Leu); 0.90 (t, J=7.3Hz, 3H, CH₃—CH₂−20); 1.26 (t, J=7.6Hz, 3H, CH₃—CH₂−7); 1.45 (m, 3H, β+β′+γ-Leu); 2.11 (q, J=7.3Hz, 2H. CH₃—CH₂−20); 2.56 (dd, J=13.2, 8.2Hz, 1H, β-Phe); 2.88 (dd, J=13.2, 4.4 Hz, 1H, β′-Phe); 3.05 (q, J=7.6Hz, 2H, CH₃—CH₂−7); 3.37 (dd, J=8.2, 4.4Hz, 1H, α-Phe); 3.99 (dd, J=18.0, 5.7Hz, 1H, α-Gly); 4.14 (dd, J=18.0, 5.7Hz, 1H, α′-Gly); 4.35 (m, 1H, α-Leu); 5.24 (s. 2H, CH₂−5); 5.47 (s, 2H, CH₂−17); 7.02 (s, 1H, H−14); 7.05-7.45 (m, 7H, H−9 +H−11+Ar-Phe); 7,93 (d, J=8.6Hz, 1H, NH-Leu); 7.99 (d, J=9.8Hz, 1H, H−12); 8.45 (t, J=5.7Hz, 1H, NH-Gly).

[0037] Example 2.

[0038] Preparation of:7-ethyl- 10-hydroxy-20-O-(L-phenylalanyl-L-leucyl-glycyl)camptothecin hydrochloride (5d; H[S]=H-Phe-Leu-Gly-, Z=H)

[0039] 7-ethyl-10-hydroxy-20-O-(L-phenylalanyl-L-leucyl-glycyl)camptothecin (5c, 0.5g), prepared as described in Example 1, was dissolved in methylene chloride (10 ml), cooled at 0° C. and treated with a 10N methanolic solution of anhydrous hydrochloric (0.2 ml). After one minute, ethyl ether (100ml) was added to the solution. The precipitate was collected, washed with ethylene chloride and dried to give 0.5g of the title compound (5d)

[0040] Example 3.

[0041] Preparation of : copolymer of N-(2-hydroxypropvlmethacrvlamide { 7-ethyl-10-hydroxy-20-O-[N-methaeryloyl-glycyl-L-phenylalanyl-L-leugyl-glycyl]camptothecin} and N-(2-hydroxypropyl) methacrvlovlglycinamide (A1)

[0042] Polymeric precursor (B1) (R₂=p-nitrophenyloxy, 2.58g, containing 1.16×10⁻³ eq. of p-nitro phenyl), prepared as described in Makromol.Chem.178, 2159 (1977), was dissolved with DMSO dry (15ml) and added with 7-ethyl-10-hydroxy-20-O-(L-phenylalanyl-L-leucyl glycyl)camptothecin (5c; 0.71g, 1 mmol). The reaction mixture was kept at room temperature for 22 hours under stirring, then 2-propanolamine (0.05ml) was added and the mixture left under stirring for one more hour. After that, the reaction mixture was precipitated with ethyl acetate (200ml) and left under stirring for 30 min. The solid material was collected on a sintered glass funnel, washed with ethyl acetate (200ml) and ethyl ether (100ml) and then dissolved with ethanol (30ml). The alcoholic solution was treated with wet DOWEX-50, sulphonic form, (1.2g) under stirring for 30min. and, after that, was added dropwise to n-hexane (200ml). The precipitate was collected on a sintered glass funnel, washed with ethyl ether and dried to constant weight to give 2.8g of the title compound (A1).

[0043] Weight-average molecular weight (Mw): 24.300.

[0044] Polydispersity (Mw/Mn): 1.63.

[0045] Content of 7-ethyl-10-hydroxy-camptothecin, determined after alkaline hydrolysis, 10% w/w.

[0046] Antitumor Activity

[0047] Compound A1 was tested on human colon carcinoma (HT29) transplanted in nude mice, in comparison with the free drug 7-ethyl-10-hydroxycamptothecin (1a) by i.v. route. A1 was found non toxic at all tested doses and gave 98% tumor inhibition at the highest tested dose of 20mg/kg (Table 1). TABLE 1 Antitumor Activity of A1 on human colon carcinoma (HT29) in comparison with 7-ethyl-10-hydroxycamptothecin (1a). treatment Dose Total Dose TI % compound schedule mg/kg mg/kg 46^(th) day Tox A1 iv q4d × 8 5 40 77 0/7 10 80 91 0/7 20 160 98 0/7 1a iv q4d × 6 20 120 97 1/7 Reference

[0048] Tumor fragment were implanted sc. Treatment started when tumor was palpable. TI% (tumor inhibition %) was calculated at day 46. 

1. A polymeric conjugate which consists essentially of: (i) from 85 to 97 mol % of N-(2-hydroxypropyl)methacryloylamide units represented by formula (2)

(ii) from 3 to 15 mol % of units represented by formula (3)

wherein [S] is a spacer group; Z is hydrogen, R₁CO in which R₁ is C₁—C₆ alkyl or the group 1,4′-bipiperidine and (iii) from 0 to 12 mol % of N-methacryloyl-glycine or N-(2-hydroxypropyl)methacryloyl-glycinamide units represented by formula (4)

wherein [X] represents hydroxy or a residue of formula —NH—CH₂—CH(OH)—CH₃.
 2. A polymeric conjugate according to claim 1 which contains the N-(2-hydroxypropyl) methacryloylamide units represented by the formula (2) in a molar proportion of 90%.
 3. A polymeric conjugate according to claim 1 which contains 10 mo% of the 20-O-(methacryloyl-glycyl-peptidyl)camptothecin units represented by the formula (3).
 4. A polymeric conjugate according to claim 1 wherein the spacer group [S] in the units of the formula (3) is selected from Ala-Gly, Phe-Gly, Leu-Gly, Phe-Ala, Leu-Leu, Phe-Leu-Gly, Leu-Leu-Gly and Phe-Leu-Gly-Gly.
 5. A polymeric conjugate according to claim 1 wherein Z in the units of the formula (3) is H.
 6. A polymeric conjugate according to claim 1 in which the content of active camptothecin derivative of formula (1)

wherein Z is as defined in claim 1, is 10% (w/w).
 7. A process for preparing a polymeric conjugate as defined in claim 1, which process comprises reacting a camptothecin derivative of formula (5):

wherein [S] and Z are as defined in claim 1, with a polymer (B) consisting essentially of: (i) from 85 to 97 mol % of N-(2-hydroxypropyl)methacryloylamide units represented by formula(2)

(iv) from 3 to 15 mol % of N-methacryloyl-glycyl units represented by formula(6)

wherein R₂is: the residue of an active ester, or hydroxy; and optionally displacing the remaining active ester groups with 1-amino-2-propanol.
 8. A ²⁰-O-peptidyl-camptothecin derivative of formula (5′)

wherein the group [S′] is a peptidyl spacer and Z is as defined in claim 1, and their salt derivatives.
 9. A process for preparing a compound of formula (5′) as defined in claim 8, which process comprises condensing a camptothecin derivative of formula 1 as defined in claim 6 with a N-protected-peptidyl derivative of formula (7): R ₃—[S′]—P  (7) wherein [S′] is as defined in claim 8, R₃ represents an amino-protecting group and P is a residue of an activated ester, to give a compound represented by formula (8):

wherein Z′ is R₃[S′] or R₁CO, wherein R₁, R₃ and [S′] are as defined above and then deblocking the N-protecting group of the substituent at position C-20 from the resulting compound.
 10. A pharmaceutical composition comprising a pharmaceutically acceptable diluent or carrier and, as active ingredient, a polymeric conjugate as defined in any one of claims 1 to 6 or a compound of formula (5′) as defined in claim
 8. 